High efficiency, large field scanning microscope

ABSTRACT

A fluorescent optical imaging system ( 10 ) produces two separate spots (S 1  and S 2 ) on a sample ( 12 ) by a pair of excitation laser beams (B 1  and B 2 ) that are generated by first and second lasers (L 1  and L 2 ). Excitation laser beams (B 1  and B 2 ) pass at slightly different angles, first through an aperture ( 15 ) of a 45° fold mirror ( 13 ), and then through an objective element ( 14 ). As a result, emission light beams ( 16, 18 ) are generated from each illuminated spot (S 1  and S 2 ) and are reflected and redirected by mirror ( 13 ) through a secondary lens ( 19 ) before reaching one of two detectors (PMT  1  and PMT  2 ). Emission beam ( 16 ) reflects off a second 45° mirror ( 22 ) prior to reaching detector (PMT  1 ), while emission beam ( 18 ) travels directly to (PMT  2 ). If desired, optical separation elements ( 24 ), such as dichroic filters, prisms, or gratings, can be positioned in front of each detector (PMT  1  and PMT  2 ). Fluorescent optical imaging system ( 10 ) may employ a scanning system ( 17 ) for illuminating and imaging the entire area of sample ( 12 ).

RELATED APPLICATION

[0001] This application is a divisional of application Ser. No.09/479,310, filed on Jan. 6, 2000.

TECHNICAL FIELD

[0002] The present invention pertains to fluorescent optical imagingsystems and, more particularly, to a non-confocal fluorescence imagingsystem for broad scale imaging of relatively large samples.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to the simultaneous imaging of twoor more fluorescently-labeled samples in a scanning optical microscope.The field of view obtained with this system is substantially larger thanconventional fluorescence microscopes, in which the field of view istypically limited by the optical design of the objective lens. Thisinvention can be applied to, but not limited to, samples such as DNAmicroarrays or tissue microarrays, where short depth of focus is notrequired, and, in fact, would degrade system performance (Cheung, V. G.,M. Morley, F. Aguilar, A. Massimi, R. Kucherlapati and G. Childs,“Making and reading microarrays,” Nature Genetics Supplement 21:15-19(1999)). It is also suitable for samples that implement fluorescentlabels with small Stokes shifts and/or overlapping absorption andemission spectra.

[0004] Difficulties can arise in fluorescence microscopy when imagingmultiple fluors with close spectral properties. It can be impractical toexcite only one fluor with a source (e.g. laser) beam due to the overlapof absorption spectra or the spectral bandwidth of the source. Thespectral emission ranges from multiple fluors may overlap, making itdifficult to direct the emission from each fluor efficiently to a singledetector, without crosstalk. Even if the emission ranges don't overlap,they may be close enough to make it difficult to obtain an effectiveoptical component (e.g. filter, grating, or prism) for separating them.One solution is to scan each wavelength independently, and then assemblea composite image from multiple scans. However, speed and imageregistration become issues in this case.

[0005] U.S. Pat. No. 5,304,810 of Amos discloses a scanning confocalmicroscope where two or more source beams with different angularorientations illuminate two distinct spots on a sample located in theobject plane of a microscope objective. The resulting reflected orfluorescent light is detected by an equal number of spaced detectors,each one receiving light from a single illuminated spot. With thissystem, the region from which light is collected by each detector (its“field of view”) is spatially limited to nearly the same area as theexcitation spot size.

[0006] An advantage of the system of Amos is that it achieves highspatial resolution at each distinct point illuminated on the specimen,which for many imaging applications is highly desirable. However, forother applications, a lower resolution image suffices.

[0007] Shalon, D., S. Smith and P. O. Brown, “A DNA micro-array systemfor analyzing complex DNA samples using two-color fluorescent probehybridization,” Genome Research 6:639-645 (1996) describe a scanner fordual wavelength fluorescence detection of DNA microarrays thatilluminates sizable spots on the sample. This is accomplished byintentionally underfilling the objective entrance pupil (i.e. the backaperture), which, by reducing the numerical aperture (NA) of theconverging beam, increases the diffraction limited spot size in thefocal plane. Note that substantially underfilling the objective aperturewith a single-transverse-mode laser beam likely results in a Gaussianintensity distribution in the focal plane, whereas overfilling theobjective aperture, as is often done in laser scanning microscopy,produces a distribution in the focal plane that approaches an Airyfunction.

[0008] As is well known in the field, it is possible to improve theaxial resolution (reduce the depth of focus) of an optical microscope byimplementing it as a confocal design. The essential benefit of aconfocal microscope is the rejection of light from out-of-focus planes,allowing imaging of thick samples without blurring (Corle, T and G.Kino, Confocal Scanning Optical Microscopy and Related Imaging Systems,Academic Press, San Diego 1996). Cheung et al. (1999) observed that aconfocal configuration actually reduced the signal-to-noise ratio, andwas therefore not beneficial, in scanning microarrays. Furthermore, thedepth-of-focus produced in a high numerical aperture confocal system issubstantially less than the typical flatness of a microscope slide. Thiscan also be an issue in a non-confocal high NA system, but is morereadily overcome. For example, in the present invention low NA sourcebeams are combined with large area detectors to reduce the sensitivityto defocus.

[0009] U.S. Pat. No. 5,459,325 of Heuton and Van Gelder discloses ahigh-speed fluorescence scanner that implements a light weight scan headcontaining a lens and mirror. This design has the advantage of variablefield of view. However, it relies on a spectral dispersion device forseparating the excitation and emission beams. As discussed above, thereare practical obstacles to spectral beamsplitting that limit itsflexibility in some applications.

[0010] Thus, an efficient, multi-wavelength scanning system formeasurement of samples that do not benefit from strict depthdiscrimination is needed. Furthermore, it should overcome thelimitations of spectral beamsplitting to allow free use of availablefluors. The present invention is directed at providing a solution tothis problem.

DISCLOSURE OF THE INVENTION

[0011] The fluorescent optical imaging system of the present invention,originally designed for the purpose of imaging hybridized DNA chips, hasa wide range of potential capabilities. A first aspect of the imagingsystem of the present invention comprises an optical source forgenerating at least two excitation beams with spatial separation forilluminating on a sample at least two distinct illuminated spots thatare spaced apart a predetermined distance, with the illuminated spotsgenerating at least two emission beams spatially or angularly separated,a detector for receiving each emission beam, and an objective elementfor directing the excitation beams onto the sample. Each detector has afield of view (receives light from a region) on the sample that islarger than an illuminated spot, but encompasses only a singleilluminated spot.

[0012] According to an aspect of the invention, the objective elementincludes a scanning mechanism for directing the excitation beams onto anarea of the sample. Preferably, the scanning mechanism includes meansfor moving the objective element in a first direction. With thisembodiment, the system further comprises means for moving the sample ina second, typically perpendicular direction. Data processing controlsand suitable imaging techniques are used to create an image of a scannedsample.

[0013] According to another aspect of the invention, the optical sourceand the objective element generate the illuminated spots in a mannercreating spots that are relatively large spots as compared todiffraction limited spots of a moderate to high numerical aperture (NA)microscope objective, such as typically used in a confocal microscope.This is an important feature of one aspect of the invention, and isdiscussed in more detail herein.

[0014] According to another aspect of the invention, there is spatialseparation of the two excitation beams. Preferably, the excitation beamsare angularly offset with respect to each other. In addition, the systemfurther comprises means for spatially separating the emission beams andredirecting the emission beams, each towards their own respectivedetector. Spatial separation of the excitation and emission beams isachieved, preferably, by means of a mirror with a small optical hole.However, other designs are possible, such as a small mirror that issmaller than an emission beam, or a prism.

[0015] According to another aspect of the invention, each detector isdisplaced from a focal point of its respective emission beam. Thisprovides a degree of de-focus, which allows for broader imagingtechniques, as discussed herein.

[0016] A second aspect of the imaging system of the present inventioncomprises an optical source for generating an excitation beam to bedirected at a sample to be imaged in a manner generating an emissionbeam from the sample, a detector for receiving the emission beam fromthe sample, an objective element between the optical source and thesample for directing the excitation beam onto the sample and forreceiving the emission beam from the sample in a manner where theexcitation beam and emission beam at least partially occupy the samespace, and an optical element for geometrically separating theexcitation beam from the emission beam and directing the emission beamtowards the detector. At the point of separation of the two beams, theexcitation beam partially occupies the emission beam.

[0017] According to an aspect of this embodiment of the imaging system,the excitation beam occupies a part of the objective element and theemission beam occupies substantially all of the objective element.Preferably, the objective element is a lens, however, a parabolic mirrorcould also be used, as well as a number of other dioptric, catoptic, andcatadioptric imaging systems.

[0018] According to another aspect of the invention, the optical elementincludes a mirror with a small hole. Alternative designs for the opticalelement, also referred to as a beamsplitter herein, include a smallmirror that is smaller than an emission beam, a prism, and several otherdesigns as described below.

[0019] According to another aspect of the invention, the excitation beamoccupies a small percentage of the space occupied by the emission beam.

[0020] According to yet another aspect of the invention, the opticalsource is adapted to generate first and second excitation beams to bedirected by the objective element toward the sample in a mannergenerating first and second emission beams. Preferably, the first andsecond excitation beams are angularly displaced from each other.Alternatively, however, the first and second excitation beams may beparallel to each other. For this alternative design, the objectiveelement may include first and second objective lenses, one for eachexcitation beam.

[0021] These and other features and advantages of the present inventionwill become more apparent from the following detailed description of theinvention, when read in conjunction with the drawings and the claims,which are all incorporated herein as part of the disclosure of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a schematic diagram of the optical imaging system of thepresent invention;

[0023]FIG. 2 is a schematic diagram contrasting the excitation spot sizeand emission field of view of the optical imaging system of FIG. 1;

[0024]FIG. 3 is a schematic diagram of a first practical embodiment ofthe optical imaging system of FIG. 1;

[0025]FIG. 4 is a schematic diagram like FIG. 3 of a second practicalembodiment where the mirror and objective lens are replaced with aparabolic mirror;

[0026]FIG. 5 is a schematic embodiment of another alternative embodimentof a parallel, dual-beam imaging system;

[0027]FIG. 6 is an enlarged schematic diagram of the beamsplitter ofFIG. 1;

[0028] FIGS. 7-13 are alternative designs for beamsplitters.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Reference will now be made in detail to the preferred embodimentsof the invention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that the describedembodiments are not intended to limit the invention specifically tothose embodiments. On the contrary, the invention is intended to coveralternatives, modifications and equivalents, which may be includedwithin the spirit and scope of the invention as defined by the appendedclaims.

[0030] The disclosure herein is intended to present a generaldescription of the system, discussing such attributes as field-of-view,resolution, magnification, radiometric efficiency, and imaging modes.Several potential variations of the system are described.

[0031] 1. System Description

[0032] Referring to FIG. 1, the fluorescent optical imaging system 10 ofthe present invention is disclosed herein for use in a scanning opticalmicroscope, but has broader applications, as discussed later. Twoseparate spots S₁ and S₂ are illuminated on a sample 12 by a pair ofexcitation laser beams B₁ and B₂ that are generated by first and secondlasers L₁ and L₂ Each of the laser sources L₁ and L₂ actually containsseveral components (e.g. a frequency doubling crystal to obtain the 532nm beam). Excitation laser beams B₁ and B₂ pass at slightly differentangles, first through an aperture 15 of a 45° fold mirror 13, and thenthrough an objective element 14, shown in FIG. 1 as a lens. As a result,emission light beams 16, 18 are generated from each illuminated spot S₁and S₂ and are reflected and redirected by mirror 13 through a secondarylens 19 before reaching one of two detectors PMT 1 and PMT 2. Emissionbeam 16 reflects off a second 45° mirror 22 prior to reaching detectorPMT 1, while emission beam 18 travels directly to PMT 2. If desired,optical separation elements 24, such as dichroic filters, prisms, orgratings, can be positioned in front of each detector PMT 1 and PMT 2.It is understood that the optimum relative positions of opticalseparation elements 24 and their respective detectors, PMT 1 and PMT 2,may be somewhat different than shown in FIG. 1.

[0033] Fluorescent optical imaging system 10 may employ a scanningsystem, indicated in phantom generally by reference numeral 17, forilluminating and imaging the entire area of sample 12. Preferably,scanning system 17 uses an objective lens scanner for one axis and asample (stage) scanner for the other, orthogonal axis. Scanning system17 is discussed in more detail later with reference to FIG. 3.

[0034] A variety of techniques may be employed for generating an angularoffset as indicated by angle θ for excitation beams B₁ and B₂. One ofbeams B₁ and B₂ may be directed through a 45° fold mirror 20 such thatbeams B₁ and B₂ enter the optical scanning system with a slight angularoffset between their propagation directions. With this design, theexcitation beams themselves are collimated or nearly collimated.

[0035] The size of the illuminated region of spots S₁ and S₂ on sample12 is determined by the excitation beam diameters, the focal length F₁of the objective lens 14, and the degree of defocus of the sample.Preferably, the excitation beam diameters are much smaller than theentrance pupil of the objective lens. The entrance pupil is the image ofthe aperture stop as seen from the source and detectors (rather thansample) side of the lens.

[0036] Objective element 14 is an aspheric singlet lens. The nominalsample focus is midway between the foci of the two excitation beams (thefoci being separated axially due to axial chromatic aberration). In thecurrent implementation, the illumination spots S₁ and S₂ are on theorder of 5-10 μm in diameter. The desired spot size and axial separationcan be adjusted by causing one or both of the excitation beams B₁ and B₂to be slightly converging or diverging, rather than collimated. Thelateral offset between the two spots is set by the angle θ of theexcitation beams and the focal length of the objective lens. Asdiscussed later, objective element 14 may alternatively include otherdesigns.

[0037] Fluorescent optical imaging system 10 employs two detectors PMT 1and PMT 2, each collecting light emitted from one of the two illuminatedspots S₁ and S₂. Detectors PMT 1 and PMT 2 are prevented from receivinglight emitted from the incorrect illumination spots by 45° mirror 22that redirects emission beam 16, effectively splitting the sample intotwo regions, each “seen” by a single detector. Detectors PMT 1 and PMT 2may include PMT, photodiode, avalanche photodiode, CCD, and otheroptical detectors.

[0038] Referring to FIG. 2, each detector “sees” one of two (or more inthe general case) non-overlapping regions R₁ and R₂ of the sample, witha border 25 between these regions determined by the position of mirror22 relative to secondary lens 19, as shown in FIG. 1. In a preferredembodiment, excitation spots S₁ and S₂ are on the order of 5-10 μm indiameter. The size of these spots is determined by the diameters andwavelengths of the incident laser beams B₁ and B₂ and the fact thatthese beams do not fill the full aperture of objective lens 14.Preferably, the distance between these spots is approximately 70 μm. Incontrast, for a confocal microscope, a typical excitation spot size on asample is on the order of 1 μm or less, due to the fact that the spot isdetermined by the diffraction limited spot size of a moderate to high NAmicroscope objective lens.

[0039] To obtain the desired size of the illuminated spots S₁, and S₂ inthe current invention, the entrance pupil of objective lens 14 isunderfilled by directing laser beams B₁ and B₂ to the lens directly,rather than expanding them to the diameter of the entrance pupil. Thisarrangement avoids use of a dichroic mirror to separate excitation lightand emission light, as is commonly done with confocal microscopes andother optical scanning systems. Instead, a 100% (ideally) reflectivemirror 13 with a 1 mm hole 15 at its center is provided. The hole couldbe circular or elliptical or a slit. The excitation light beams(typically 0.8 mm in diameter) pass through the hole, while fluorescentemission light reflects from the mirror and passes through a barrierfilter, if necessary, to remove scattered excitation light and throughsecondary lens 19 to the light detectors PMT 1 and PMT 2.

[0040] The field of view of detectors PMT 1 and PMT 2 also differs fromthe field of view of the detectors in a typical confocal microscope.Note that field of view in this context refers to the image of thedetector or detector aperture on the sample, not the total size of thescanned region. As shown in FIG. 2, the complete field of view of thedetectors in the present invention is that of region 26, whichencompasses both excitation spots S₁ and S₂. While shown as a circle inFIG. 2, it is understood that region 26 may have other shapes. Forexample, the field of view may be 200 mm across at sample 12. Mirror 22placed before detectors PMT 1 and PMT 2 splits the field of view intotwo halves 28, 30 so that each detector “sees” approximately half of thefull field of view. In contrast, the field of view of a confocalmicroscope is spatially limited to 1 mm or less, nearly the same size asthe excitation spot, which is necessary to maximize the lateral higherresolution. The field of view in a confocal microscope is typicallylimited by an aperture placed in front of the detector.

[0041] One additional difference between the present system and aconfocal system has to do with the depth of field (axial resolution). Ina typical high-magnification confocal microscope, the use of a detectoraperture to reject out-of-focus light limits the depths of field to 1 mmor less. Since the optical system of the present invention does notactively reject out of focus light with detector apertures or smalldetectors, the depth of field is significantly greater than a confocalmicroscope.

[0042] As with all scanning optical microscopes, imaging system 10creates an image by sequentially acquiring pixel data (e.g.fluorescence) and constructing an image with known computer graphicstechniques. With two excitation beams having a small lateral offsetbetween their test locations on the sample, the present system forms twocomplete images with a known lateral offset between them. Scanning isperformed in the two lateral directions.

[0043] In a first practical embodiment of the system, as shown in FIG.3, there are two scanning mechanisms employed, each in the form of atranslation stage 40, 42. Translation stage 40 is accompanied by alinear encoder assembly 43. In a fast-scan direction, as indicated byarrow 44, the excitation beams B₁ and B₂ enter the scanner stage area inan approximately horizontal orientation, are reflected up towards asample 12 by a fold mirror 46, and are then focused by objective lens 14onto sample 12. Movement of translation stage 40 in the direction ofarrow 44 provides movement in a first direction, where the measurementlocation on the sample is determined by the optical axis of objectiveelement 14. The slow-axis scan is implemented by moving sample 12 viatranslation stage 42 in a second direction orthogonal to the first scandirection, which second direction is into and out of the page as shown.

[0044]FIG. 3 also illustrates an alternative two-wavelength version ofthe imaging system of the present invention. Additional dichroicbeamsplitters 44, 50 and bandpass filters 54, 56 are used to mix lightfrom several lasers together (two lasers shown) and to separatefluorescent emission light to several bands. This arrangement can beused for simultaneous scanning with multiple source and detectionwavelengths. As discussed below regarding the beamsplitter shown in FIG.10, the performance of this design can be limited by the characteristicsof the dichroic beamsplitters.

[0045] 2. Alternate Design Forms

[0046] Slight variations to the design of the present system can make ita general purpose microscope or can tailor it for specific applications.These variations include the beam source or sources, beamsplitting,detection, and scanning attributes, as discussed herein.

[0047] A) Beam Sources

[0048] A number of alternate excitation light source configurations canbe implemented in this system. The simplest case is the use of a singleon-axis laser beam. The sample location will be at the focal point ofthe objective lens (assuming a collimated laser beam). The portion ofthe lens through which the beam travels does not vary with scanposition. This is somewhat simpler than the general implementation shownin FIG. 1, in which the finite angles the two excitation beams make withthe optical axis causes them to move towards and away from the center ofthe objective lens during each scan of the lens. This produces slightlyvarying illumination conditions throughout the scan, something that doesnot occur with beams coaxial with the lens axis. It is also possible touse two or more coaxial source beams. In this case, they will illuminatethe same spot on the sample (neglecting chromatic aberration).

[0049] It is also possible to obtain separate illumination spots byusing two parallel beams, each of which illuminates a portion of theobjective lens aperture. The individual beams converge as separate conesof light, and are coincident in the focal plane of the lens (assumingcollimated source beams incident on the objective lens). A disadvantageof this technique compared to the angled beams is that the separationbetween the two converging beams varies with defocus of the sample, andwill be impacted by vertical runout (defocus) during the scan. As thesample comes closer to the focus of the lens, the two illumination spotswill approach each other. In the angular offset case of the currentdesign, this effect is much less pronounced (the two spots are nevercoincident, assuming the two beams cross in front of the objective lensrather than between the lens and sample). The effect of runout onseparation between the illumination spots can be minimized in thecurrent invention by causing the two illumination beams to cross at ornear the front focal point of the objective lens.

[0050] Multiple beams may come from one or more sources, and may or maynot have different spectral characteristics. It is also possible tobring one or both of the source beams in from above the sample (oppositethe objective lens). This is discussed below in the trans-illuminationsection of alternate imaging modes.

[0051] Smaller illumination spots may be obtained by increasing thediameter of the source beams (e.g. by passing the beams through aspatial filter/beam expander) and overfilling the entrance pupil of theobjective lens. The spatial filter consists of a positive lens to focusthe beam and a pinhole intended to pass only the central lobe of thediffraction (Airy) pattern. The beam expands beyond the pinhole and iscollimated by another lens. This beam is then focused on the sample bythe objective lens, producing a diffraction limited illumination spot.In this implementation, the illumination spots produced by two angledsource beams will be separated only in the immediate region of thefocus, and a well-corrected objective lens may be required to obtainadequate performance.

[0052]FIG. 4 illustrates a variation on the design of FIG. 3. In thisembodiment, a single off-axis parabolic mirror 60 is used in place ofthe combination objective lens and 45° mirror. Parabolic mirror 60performs multiple functions.

[0053] It turns the axis of the incoming laser beams B₁ and B₂ by 90°;it focuses those beams into waists coinciding with the sample surface12; it collects and recollimates fluorescence emissions, and turns theemissions beams by 90°.

[0054]FIG. 5 illustrates a split, parallel dual beam system. Excitationlight beams B₁ and B₂ are generated by separate lasers L₁ and L₂ in aparallel orientation. A mirror 64 with two holes 66, 68 is provided inthe path of excitation beams B₁ and B₂ to direct the beams at twoadjacent objective lenses 70, 72. A pair of illuminated spots S₁ and S₂are generated, fluorescence emissions from which are collimated byobjective lenses 70, 72 as emission beams 76, 78. Mirror 64 redirectsemission beams 76, 78 in a spatially separated manner toward detectorsPMT 1 and PMT 2, with a 45° mirror 80 provided for emission beam 78.This optical layout removes the need for a secondary lens, although onecan be included if beneficial.

[0055] B) Beamsplitter

[0056] The optical system of the present invention utilizes a noveldesign for a beamsplitter, which in FIG. 1 is illustrated in the form ofa mirror with a small hole. An enlarged illustration of this design inshown in FIG. 6. Excitation beams B₁ and B₂ (two in the illustratedsystem, but one or more in the general case) pass through hole 15 in thecenter of mirror 13. The objective side of mirror 13 includes areflective coating 84 for redirecting emission light beams 16, 18.

[0057] The fluorescent emission light 16, 18 emitted from the samplereturns along the same direction, but with a much larger diameter thanexcitation beams B₁ and B₂. Specifically, excitation beams B₁ and B₂have a smaller diameter than the entrance pupil of the objective lens,while the collected emission light 16, 18 has the diameter of theobjective pupil (assuming collimated emission beams). Due to the largedisparity between the diameters of the excitation and emission beams,only a small amount of emission light is lost through the hole 15 in thecenter of mirror 13, making this an effective beamsplitter.

[0058] Beams B₁ and B₂ in FIG. 6, as well as in FIGS. 7-13, are shown tobe coaxial for illustrative purposes. They may or may not be coaxial inpractice.

[0059] As shown in FIG. 7, the hole in the center of the mirror can becreated by placing a reflective coating 86 on one surface of an opticalwindow 88, leaving a small region 90 in the center uncoated (oranti-reflective coated).

[0060] The reflective coating may be replaced by a totally internallyreflecting surface, as shown in FIG. 8. In this case, a small prism 85and a large prism 87 are combined such that the illumination beams B₁,B₂ enter the small prism, passes through the interface between theprisms, and exits the large prism. The emission beams 16, 18, which havea much larger diameter than the illumination beam, enter large prism 87but do not pass through to small prism 85 except where the two prismsare in contact. Instead, the majority of the beam is totally internallyreflected and exits the other face of the large right-angle prism.

[0061] Another example is shown in FIG. 9, where a transparent mirrorsubstrate 90 (optical window) has a partially reflective coating 92 onone surface. In this embodiment, a portion of emission beams 16′ and 18′travel back through the optical window 90.

[0062] In this design, the partially reflective coating causes a portion(e.g., 50%) of any beam impinging on it to be reflected, and theremaining portion transmitted (neglecting absorption). This is a commondesign for comparable illumination (excitation) and detection (emission)beam diameters. However, it suffers from a minimum of 75% loss (theillumination beam is transmitted through the beamsplitter and thedetection beam reflected off it, or vice-versa). In the present system,where the excitation beam is substantially smaller in diameter than theemission beam, this loss is unnecessarily high and for this reason isnot a preferred arrangement. A plate beamsplitter is shown in FIG. 9,but this type of beamsplitter can also be implemented in other forms,such as a cube or a pellicle.

[0063] A variation on the design shown in FIG. 9 employs a polarizingbeamsplitter and quarter-wave plate to improve the efficiency of thesystem. In this case, the beamsplitter reflects linearly polarizedlight, which is then converted to circularly polarized light by thequarter-wave plate. Upon reflection from the sample, the return beam isconverted by the quarter-wave plate back into linearly polarized light,but now with the correct polarization to be transmitted by thepolarizing beamsplitter. The order of reflection and transmission by thepolarizing beamsplitter may be reversed. This polarization technique istypically used in reflection, rather than fluorescence, due to thepreservation of polarization.

[0064] Yet another beamsplitter example is shown in FIG. 10, where adichroic beamsplitter 94 is employed having a dichroic coating 96. Thedichroic beamsplitter design utilizes a beamsplitter coating thatreflects (or transmits) one or more wavelength ranges while transmitting(reflecting) other wavelength ranges. This is an efficient solution influorescence operation where the illumination wavelengths and emissionwavelengths are spectrally shifted from one another. However, thepractical limitations of thin film coatings and the overlap betweenabsorption and emission spectra of fluorescent dyes prevent this frombeing a perfect solution in fluorescence microscopy.

[0065] Yet still another example of a beamsplitter is shown if FIG. 11.A small mirror 95 reflects excitation beams B₁ and B₂ while emissionbeams 16, 18 pass primarily around the mirror. FIG. 11 shows essentiallythe reciprocal case of FIG. 6. Rather than transmitting the excitationbeam(s) through a small hole in a mirror that reflects the detectionbeam, as is done with the beamsplitter of FIG. 6, the beams arereflected from a small mirror 95, while emission beams 16, 18 aretransmitted around the mirror. The small mirror can also be implementedby placing a reflective coating on only a small region of an opticalwindow, with the rest of the window left uncoated (or with ananti-reflection coating). Alternatively, the mirror could collimate (orotherwise image) the excitation beams, as well as reflecting them. Thismay be suitable for a fiber source brought in from the side of the beam.

[0066]FIG. 12 shows still another example where a fiber source 99 with alens 98 directs a collimated excitation beam B₁ at the objectiveelement. Emission beam 16 passes around lens 98 and fiber source 99towards the detector.

[0067] It is also possible to add focusing power to the beamsplitter. InFIG. 13, an off-axis parabolic mirror segment 100 with a hole 102 in itscenter redirects emission beams 16, 18, focusing them at the detectors.The parabolic mirror shown in FIG. 13 provides a way to combine theexcitation and emission beams, as is done by the beamsplitter of FIG. 6,but also focuses the emission beam. The imaging lens used to focus thelight onto the detectors can now be omitted, its job performed by theparabolic mirror. A parabolic mirror provides perfect (aberration-free)imaging for a collimated, on-axis, input beam. However, the imagingperformance of the parabolic mirror degrades quickly for off-axisimaging, and various shaped lenses may be preferred in certain cases.

[0068] C) Detectors

[0069] A single detector is currently employed to collect light fromeach illuminated region of the sample. Two or more detectors may be usedto collect light from each spot with, for example, spectral or spatialdifferences between the light received by the detectors. The emissionfrom the sample may be separated into two or more spectral regions with,for example, a dichroic beamsplitter, a dispersive element such as aprism, or a diffractive element such as a grating.

[0070] The preferred detection scheme uses a mirror to divert light fromone of the two illuminated spots to one of the detectors, while allowinglight from the other illuminated spot to pass undisturbed to the otherdetector. In a sense, the edge of this mirror is imaged onto the sample,separating it into two non-overlapping “detection” regions.

[0071] It is possible reduce the size of these detection regions byplacing apertures in front of one or more of the detectors. Inclusion ofsuch apertures does not convert the present system to a confocalmicroscope. In a confocal microscope, three-dimensional imaging ispossible due to the rejection of light from out-of-focus regions of thesample. This is accomplished by simultaneous diffraction limited imagingof a point source and a point detector onto the same point on thesample; these conditions are not met in the current system.

[0072] 4. Imaging Modes

[0073] While the present system is nominally designed forepi-illumination fluorescence imaging, a number of other operating modesare possible. These modes include, but are not limited to,trans-illumination (transmission), darkfield, brightfield, confocal,interferometric, polarization, and differential interference contrast(DIC).

[0074] In an epi-illumination system, the source beams illuminate thesample through the same lens as is used to collect light for detection.The present system can be modified to a trans-illumination system bybringing in the illumination from above the sample, while the objectivelens collects the resulting fluorescent light from below the sample.Equivalently, the sample can be illuminated from below while thecollection lens is placed above the sample. In the trans-illuminationfluorescence mode, the portion of the illumination beam that istransmitted by the sample can pass through to the detectors. Separationof the source and fluorescent signals is generally accomplished withspectral filters, and imperfections in these filters (i.e. finitestopband transmission) will reduce the signal-to-noise ratio of thesystem. The present system uses a novel beamsplitter with a small holeto pass the illumination beams while reflecting the majority of thelarger diameter detection beams. In a trans-illumination system, asimilar hole can be placed in a fold mirror in the detection path toavoid passage of the transmitted source beams to the detectors.

[0075] The present system is suitable for studying the fluorescenceproperties of DNA chips, such as images with little depth resolution ofthe type that can determine which sequence of nucleotides is present insample DNA, or which genes are over- or underexpressed, simply bydetermining which area of the DNA chip containing complementary DNAsequence is fluorescent. However, the present system can be readily usedto study the scattering properties of these and other samples. In theepi-illumination mode, removal of the spectral filters in the detectionpath(s) will allow collection of light reflected from the sample(although care must be taken to ensure that the reflected source beamsreach the detectors, rather than pass through the hole in thebeamsplitter). This is also possible in trans-illumination, wheretransmission and forward scattering of the light will be measured.

[0076] Several other imaging modes are possible with modifications tothe present system. As discussed above, inclusion of small apertures infront of the detectors, in conjunction with reduced illumination spots,will produce a confocal system. This will allow imaging of thick objectsand three-dimensional imaging. Another possibility is use as aninterference microscope (a non-fluorescence implementation), where theobjective lens will need to include a beamsplitter and referencesurface. This allows three-dimensional imaging of reflective objectswith subwavelength axial resolution. Polarization and DIC imaging modesare also foreseeable, although they require additional components in thesystem.

[0077] The foregoing descriptions of specific embodiments of the presentinvention have been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise forms disclosed, and obviously manymodifications and variations are possible in light of the aboveteaching. The embodiments were chosen and described in order to bestexplain the principles of the invention and its practical application,to thereby enable others skilled in the art to best utilize theinvention and various embodiments with various modifications as aresuited to the particular use contemplated. It is intended that the scopeof the invention be defined by the claims appended hereto when read andinterpreted according to accepted legal principles such as the doctrineof equivalents and reversal of parts.

What is claimed is:
 1. A fluorescent optical imaging system comprising:an optical source for generating an excitation beam to be directed at asample to be imaged in a manner generating an emission beam from thesample; a detector for receiving the emission beam from the sample; aparabolic mirror between the optical source and the sample forreflecting the excitation beam onto the sample and for receiving theemission beam from the sample in a manner where the excitation beam andemission beam at least partially occupy the same space; and an opticalelement for geometrically separating the excitation beam from theemission beam and directing the emission beam toward the detector. 2.The fluorescent optical imaging system of claim 1 wherein the excitationbeam occupies a part of the parabolic mirror and the emission beamoccupies substantially all of the parabolic mirror.
 3. The fluorescentoptical imaging system of claim 1 wherein the optical element includes asmall mirror that is smaller than an emission beam.
 4. The fluorescentoptical imaging system of claim 1 wherein the optical element includes aprism.
 5. The fluorescent optical imaging system of claim 1 wherein theexcitation beam occupies a small percentage of the space occupied by theemission beam.
 6. The fluorescent optical imaging system of claim 1wherein the optical source is adapted to generate first and secondexcitation beams to be directed by the objective element toward thesample in a manner generating first and second emission beams.
 7. Amethod of fluorescent optical imaging comprising the steps of;generating an excitation beam to be directed at a sample to be imaged ina manner generating an emission beam from the sample; detecting theemission beam from the sample; directing the excitation beam onto aparabolic mirror and onto the sample and gathering the emission beamswith the parabolic mirror, in a manner where the excitation beam andemission beam at least partially occupy the same space; andgeometrically separating the excitation beam from the emission beam anddirecting the emission beam towards the detector.
 8. The method of claim7 wherein the excitation beam occupies a part of the parabolic mirrorand the emission beam occupies substantially all of the parabolicmirror.
 9. The method of claim 7 wherein the step of geometricallyseparating the excitation and emission beams includes use of a mirrorwith a small hole.
 10. The method of claim 8 wherein the step ofgeometrically separating the excitation and emission beams includes useof a small mirror that is smaller than an emission beam.
 11. The methodof claim 7 wherein first and second excitation beams are directed by theparabolic mirror toward the sample in a manner generating first andsecond emission beams.